The R2TP complex regulates paramyxovirus RNA synthesis.

The regulation of paramyxovirus RNA synthesis by host proteins is poorly understood. Here, we identified a novel regulation mechanism of paramyxovirus RNA synthesis by the Hsp90 co-chaperone R2TP complex. We showed that the R2TP complex interacted with the paramyxovirus polymerase L protein and that silencing of the R2TP complex led to uncontrolled upregulation of mumps virus (MuV) gene transcription but not genome replication. Regulation by the R2TP complex was critical for MuV replication and evasion of host innate immune responses. The R2TP complex also regulated measles virus (MeV) RNA synthesis, but its function was inhibitory and not beneficial to MeV, as MeV evaded host innate immune responses in the absence of the R2TP complex. The identification of the R2TP complex as a critical host factor sheds new light on the regulation of paramyxovirus RNA synthesis.

[Future perspectives of mumps vaccine].

Because of the concerns about aseptic meningitis due to Japanese domestic mumps vaccine strains, the routine mumps immunization program has not yet been introduced in Japan, and it resulted in the situation where the major mumps epidemics occur every 4-5 years. However, the fact that at least 348 mumps hearing loss cases were reported during the recent epidemic period in 2015-2016, argues that the introduction of the routine mumps immunization program is an urgent issue for us. In contrast, 122 countries employ mumps-containing vaccines for nationwide immunization programs by 2018, of which 117 apply 2-dose vaccination regimens, and many of them use Jeryl-Lynn containing measles-mumps-rubella (MMR) vaccines. While in these countries, where mumps seemed to have been controlled, mumps resurgented in the 2000s. Although, the concerns surrounding mumps vaccination are extremely different in Japan and abroad, both of them link to the inherent characteristics of mumps vaccine, in which it is hard to balance the safety and the efficacy. In order to promptly introduce the routine mumps immunization program in Japan, Japanese domestic mumps vaccine strains need to be re-evaluated based on the latest evidence. Furthermore, from a long-range viewpoint, a novel mumps vaccine should be developed, which combines the safety and the efficacy.

Heat Shock Protein 90 Ensures Efficient Mumps Virus Replication by Assisting with Viral Polymerase Complex Formation.

Paramyxoviral RNAs are synthesized by a viral RNA-dependent RNA polymerase (RdRp) consisting of the large (L) protein and its cofactor phosphoprotein (P protein). The L protein is a multifunctional protein that catalyzes RNA synthesis, mRNA capping, and mRNA polyadenylation. Growing evidence shows that the stability of several paramyxovirus L proteins is regulated by heat shock protein 90 (Hsp90). In this study, we demonstrated that Hsp90 activity was important for mumps virus (MuV) replication. The Hsp90 activity was required for L-protein stability and activity because an Hsp90-specific inhibitor, 17-allylamino-17-demethoxygeldanamycin (17-AAG), destabilized the MuV L protein and suppressed viral RNA synthesis. However, once the L protein formed a mature polymerase complex with the P protein, Hsp90 activity was no longer required for the stability and activity of the L protein. When the Hsp90 activity was inhibited, the MuV L protein was degraded through the CHIP (C terminus of Hsp70-interacting protein)-mediated proteasomal pathway. High concentrations of 17-AAG showed strong cytotoxicity to certain cell types, but combined use of an Hsp70 inhibitor, VER155008, potentiated degradation of the L protein, allowing a sufficient reduction of 17-AAG concentration to block MuV replication with minimum cytotoxicity. Regulation of the L protein by Hsp90 and Hsp70 chaperones was also demonstrated for another paramyxovirus, the measles virus. Collectively, our data show that the Hsp90/Hsp70 chaperone machinery assists in the maturation of the paramyxovirus L protein and thereby in the formation of a mature RdRp complex and efficient viral replication.IMPORTANCE Heat shock protein 90 (Hsp90) is nearly universally required for viral protein homeostasis. Here, we report that Hsp90 activity is required for efficient propagation of mumps virus (MuV). Hsp90 functions in the maintenance of the catalytic subunit of viral polymerase, the large (L) protein, prior to formation of a mature polymerase complex with the polymerase cofactor of L, phosphoprotein. Hsp70 collaborates with Hsp90 to regulate biogenesis of the MuV L protein. The functions of these chaperones on the viral polymerase may be common among paramyxoviruses because the L protein of measles virus is also similarly regulated. Our data provide important insights into the molecular mechanisms of paramyxovirus polymerase maturation as well as a basis for the development of novel antiviral drugs.

Cross-Neutralization between Human and African Bat Mumps Viruses.

Recently, a new paramyxovirus closely related to human mumps virus (MuV) was detected in bats. We generated recombinant MuVs carrying either or both of the fusion and hemagglutinin-neuraminidase bat virus glycoproteins. These viruses showed replication kinetics similar to human MuV in cultured cells and were neutralized efficiently by serum from healthy humans.

Mumps Virus Is Released from the Apical Surface of Polarized Epithelial Cells, and the Release Is Facilitated by a Rab11-Mediated Transport System.

UNLABELLED: Mumps virus (MuV) is an airborne virus that causes a systemic infection in patients. In vivo, the epithelium is a major replication site of MuV, and thus, the mode of MuV infection of epithelial cells is a subject of interest. Our data in the present study showed that MuV entered polarized epithelial cells via both the apical and basolateral surfaces, while progeny viruses were predominantly released from the apical surface. In polarized cells, intracellular transport of viral ribonucleoprotein (vRNP) complexes was dependent on Rab11-positive endosomes, and vRNP complexes were transported to the apical membrane. Expression of a dominant negative form of Rab11 (Rab11S25N) reduced the progeny virus release in polarized cells but not in nonpolarized cells. Although in this way these effects were correlated with cell polarity, Rab11S25N did not modulate the direction of virus release from the apical surface. Therefore, our data suggested that Rab11 is not a regulator of selective apical release of MuV, although it acts as an activator of virus release from polarized epithelial cells. In addition, our data and previous studies on Sendai virus, respiratory syncytial virus, and measles virus suggested that selective apical release from epithelial cells is used by many paramyxoviruses, even though they cause either a systemic infection or a local respiratory infection. IMPORTANCE: Mumps virus (MuV) is the etiological agent of mumps and causes a systemic infection. However, the precise mechanism by which MuV breaks through the epithelial barriers and achieves a systemic infection remains unclear. In the present study, we show that the entry of MuV is bipolar, while the release is predominantly from the apical surface in polarized epithelial cells. In addition, the release of progeny virus was facilitated by a Rab11-positive recycling endosome and microtubule network. Our data provide important insights into the mechanism of transmission and pathogenesis of MuV.

Heat shock protein 70 regulates degradation of the mumps virus phosphoprotein via the ubiquitin-proteasome pathway.

UNLABELLED: Mumps virus (MuV) infection induces formation of cytoplasmic inclusion bodies (IBs). Growing evidence indicates that IBs are the sites where RNA viruses synthesize their viral RNA. However, in the case of MuV infection, little is known about the viral and cellular compositions and biological functions of the IBs. In this study, pulldown purification and N-terminal amino acid sequencing revealed that stress-inducible heat shock protein 70 (Hsp72) was a binding partner of MuV phosphoprotein (P protein), which was an essential component of the IB formation. Immunofluorescence and immunoblotting analyses revealed that Hsp72 was colocalized with the P protein in the IBs, and its expression was increased during MuV infection. Knockdown of Hsp72 using small interfering RNAs (siRNAs) had little, if any, effect on viral propagation in cultured cells. Knockdown of Hsp72 caused accumulation of ubiquitinated P protein and delayed P protein degradation. These results show that Hsp72 is recruited to IBs and regulates the degradation of MuV P protein through the ubiquitin-proteasome pathway. IMPORTANCE: Formation of cytoplasmic inclusion bodies (IBs) is a common characteristic feature in mononegavirus infections. IBs are considered to be the sites of viral RNA replication and transcription. However, there have been few studies focused on host factors recruited to the IBs and their biological functions. Here, we identified stress-inducible heat shock protein 70 (Hsp72) as the first cellular partner of mumps virus (MuV) phosphoprotein (P protein), which is an essential component of the IBs and is involved in viral RNA replication/transcription. We found that the Hsp72 mobilized to the IBs promoted degradation of the MuV P protein through the ubiquitin-proteasome pathway. Our data provide new insight into the role played by IBs in mononegavirus infection.

Virus genotypes and responses of serum-specific antibodies in children with primary mumps and mumps reinfection.

BACKGROUND: Research on children with mumps reinfection after natural infection is limited; there are currently no studies on virus-specific antibody responses in paired sera or genotyping of isolated viruses. METHODS: This study included 281 children (147 boys and 134 girls, age: 1.2-15.9 y) with primary mumps (240), mumps reinfection after natural infection (9), mumps after previous vaccination (26), and vaccine-associated mumps (6). We measured mumps-specific serum antibodies and analyzed isolated virus genes. RESULTS: During acute illness, series-specific IgM and IgG titers exceeded cutoff values in 240 and 232 children with primary mumps, respectively. During convalescence, IgM antibodies were positive in seven and negative in two of nine children with mumps reinfection occurring after natural infection; among 26 previously vaccinated children, 13 were positive and 13 negative. Mumps viruses were isolated from viral cultures from 42 of the 51 children. Except for 6 vaccine-associated cases, all remaining 36 cases of isolated mumps virus were identified as genotype G. CONCLUSION: These results suggest that measurement of IgM antibody on any day of acute illness may be indicative of primary mumps but may be inconsistent for diagnosing mumps reinfection after natural infection or previous vaccination.

MicroRNA regulation of glycoprotein B5R in oncolytic vaccinia virus reduces viral pathogenicity without impairing its antitumor efficacy.

Vaccinia virus, once widely used for smallpox vaccine, has recently been engineered and used as an oncolytic virus for cancer virotherapy. Their replication has been restricted to tumors by disrupting viral genes and complementing them with products that are found specifically in tumor cells. Here, we show that microRNA (miRNA) regulation also enables tumor-specific viral replication by altering the expression of a targeted viral gene. Since the deletion of viral glycoprotein B5R not only decreases viral pathogenicity but also impairs the oncolytic activity of vaccinia virus, we used miRNA-based gene regulation to suppress B5R expression through let-7a, a miRNA that is downregulated in many tumors. The expression of B5R and the replication of miRNA-regulated vaccinia virus (MRVV) with target sequences complementary to let-7a in the 3'-untranslated region (UTR) of the B5R gene depended on the endogenous expression level of let-7a in the infected cells. Intratumoral administration of MRVV in mice with human cancer xenografts that expressed low levels of let-7a resulted in tumor-specific viral replication and significant tumor regression without side effects, which were observed in the control virus. These results demonstrate that miRNA-based gene regulation is a potentially novel and versatile platform for engineering vaccinia viruses for cancer virotherapy.

Nationwide and long-term molecular epidemiologic studies of mumps viruses that circulated in Japan between 1986 and 2017.

In Japan, major mumps outbreaks still occur every 4-5 years because of low mumps vaccine coverage (30-40%) owing to the voluntary immunization program. Herein, to prepare for a regular immunization program, we aimed to reveal the nationwide and long-term molecular epidemiological trends of the mumps virus (MuV) in Japan. Additionally, we performed whole-genome sequencing (WGS) using next-generation sequencing to assess results from conventional genotyping using MuV sequences of the small-hydrophobic (SH) gene. We analyzed 1,064 SH gene sequences from mumps clinical samples and MuV isolates collected from 25 prefectures from 1986 to 2017. The results showed that six genotypes, namely B (110), F (1), G (900), H (3), J (41), and L (9) were identified, and the dominant genotypes changed every decade in Japan since the 1980s. Genotype G has been exclusively circulating since the early 2000s. Seven clades were identified for genotype G using SH sequence-based classification. To verify the results, we performed WGS on 77 representative isolates of genotype G using NGS and phylogenetically analyzed them. Five clades were identified with high bootstrap values and designated as Japanese clade (JPC)-1, -2, -3, -4, -5. JPC-1 and -3 accounted for over 80% of the total genotype G isolates (68.3 and 13.8%, respectively). Of these, JPC-2 and -5, were newly identified clades in Japan through this study. This is the first report describing the nationwide and long-term molecular epidemiology of MuV in Japan. The results provide information about Japanese domestic genotypes, which is essential for evaluating the mumps elimination progress in Japan after the forthcoming introduction of the mumps vaccine into Japan's regular immunization program. Furthermore, the study shows that WGS analysis using NGS is more accurate than results obtained from conventional SH sequence-based classification and is a powerful tool for accurate molecular epidemiology studies.

Characterization of mumps viruses circulating in Mongolia: identification of a novel cluster of genotype H.

Although mumps virus is still causing annual epidemics in Mongolia, very few epidemiological and virological data have been reported. We describe here the first phylogenetic analysis data on the mumps viruses circulated in Mongolia in 2009. We detected 21 mumps virus cDNAs and obtained a virus isolate from 32 throat swabs of mumps patients in Ulaanbaatar, the capital of Mongolia. The phylogenetic analyses based on the 316 nucleotides of the small hydrophobic gene show that these sequences form a single cluster, with the closest relatedness to the viruses belonging to genotype H. According to the recommendation of the World Health Organization, Mongolian mumps viruses could be classified into a novel genotype because the divergence between new sequences and genotype H reference viruses is >5% (6.3 to 8.2%). However, additional analyses based on the fusion gene, the hemagglutinin-neuraminidase gene, and the whole-genome indicate that the divergences between the Mongolian isolate and other genotype H strains never exceed the within-genotype divergences of other genotypes. These results suggest that Mongolia strains should be included in genotype H and that the current criteria for mumps virus genotyping should be revised. We propose here that the Mongolian viruses should be classified as a new subgenotype termed H3. Since previous epidemiological studies suggested that genotypes H may be associated with central nervous system diseases, we evaluated the neurovirulence of the Mongolian isolate in the neonatal rat system. However, the virus does not exhibit prominent neurovirulence in rats.

Antiviral Activity of CD437 Against Mumps Virus.

Many efforts have been dedicated to the discovery of antiviral drug candidates against the mumps virus (MuV); however, no specific drug has yet been approved. The development of efficient screening methods is a key factor for the discovery of antiviral candidates. In this study, we evaluated a screening method using an Aequorea coerulescens green fluorescent protein-expressing MuV infectious molecular clone. The application of this system to screen for active compounds against MuV replication revealed that CD437, a retinoid acid receptor agonist, has anti-MuV activity. The point of antiviral action was a late step(s) in the MuV life cycle. The replication of other paramyxoviruses was also inhibited by CD437. The induction of retinoic acid-inducible gene (RIG)-I expression is a reported mechanism for the antiviral activity of retinoids, but our results indicated that CD437 did not stimulate RIG-I expression. Indeed, we observed antiviral activity despite the absence of RIG-I, suggesting that CD437 antiviral activity does not require RIG-I induction.

Prior immunization with severe acute respiratory syndrome (SARS)-associated coronavirus (SARS-CoV) nucleocapsid protein causes severe pneumonia in mice infected with SARS-CoV.

The details of the mechanism by which severe acute respiratory syndrome-associated coronavirus (SARS-CoV) causes severe pneumonia are unclear. We investigated the immune responses and pathologies of SARS-CoV-infected BALB/c mice that were immunized intradermally with recombinant vaccinia virus (VV) that expressed either the SARS-CoV spike (S) protein (LC16m8rVV-S) or simultaneously all the structural proteins, including the nucleocapsid (N), membrane (M), envelope (E), and S proteins (LC16m8rVV-NMES) 7-8 wk before intranasal SARS-CoV infection. The LC16m8rVV-NMES-immunized group exhibited as severe pneumonia as the control groups, although LC16m8rVV-NMES significantly decreased the pulmonary SARS-CoV titer to the same extent as LC16m8rVV-S. To identify the cause of the exacerbated pneumonia, BALB/c mice were immunized with recombinant VV that expressed the individual structural proteins of SARS-CoV (LC16mOrVV-N, -M, -E, -S) with or without LC16mOrVV-S (i.e., LC16mOrVV-N, LC16mOrVV-M, LC16mOrVV-E, or LC16mOrVV-S alone or LC16mOrVV-N + LC16mOrVV-S, LC16mOrVV-M + LC16mOrVV-S, or LC16mOrVV-E + LC16mOrVV-S), and infected with SARS-CoV more than 4 wk later. Both LC16mOrVV-N-immunized mice and LC16mOrVV-N + LC16mOrVV-S-immunized mice exhibited severe pneumonia. Furthermore, LC16mOrVV-N-immunized mice upon infection exhibited significant up-regulation of both Th1 (IFN-gamma, IL-2) and Th2 (IL-4, IL-5) cytokines and down-regulation of anti-inflammatory cytokines (IL-10, TGF-beta), resulting in robust infiltration of neutrophils, eosinophils, and lymphocytes into the lung, as well as thickening of the alveolar epithelium. These results suggest that an excessive host immune response against the nucleocapsid protein of SARS-CoV is involved in severe pneumonia caused by SARS-CoV infection. These findings increase our understanding of the pathogenesis of SARS.

Seroprevalence of mumps before the introduction of mumps-containing vaccine in Lao PDR: results from a nationwide cross-sectional population-based survey.

OBJECTIVE: Mumps-containing vaccine is currently not a component of the national immunization schedule in Lao People's Democratic Republic (PDR). Mumps itself is not a notifiable disease in the country and the seroprevalence of anti-mumps immunoglobulin G (IgG) in the general population is unknown. In this study, anti-mumps IgG was measured in 2058 blood samples to evaluate population immunity in the country. RESULTS: The seroprevalence of anti-mumps IgG showed a gradual increase with increasing age, starting at 10.6% (95% CI 7.4-13.7) in participants aged 1-2 years, and almost plateaued at about 75% in individuals older than 11-12 years, though it still tended toward a small increase up to 89.6% (95% CI 86.6-92.6) in participants aged 40 years or older. Compared with the results of previous studies, this increase with increasing age is less marked and the plateau of anti-mumps seroprevalence is lower. We attribute this result mainly to the lower population density in Lao PDR.

Molecular Epidemiological Study of Mumps Epidemics of 2015 in Okinawa, Japan.

Although major mumps epidemics occurred every 4-5 years in Okinawa Prefecture in Japan, no laboratory diagnoses were conducted. A mumps epidemic started in Okinawa in October 2014, and we collected clinical samples from 31 patients in 4 areas (Hokubu, Nanbu, Miyako, and Yaeyama) from July to December 2015, for virus isolation and RT-PCR, whose positive ratios were 52% and 87%, respectively. Phylogenetic analyses showed that all isolates were classified into genotype G, and with one exception, consisted of 2 subgenotypes, Ge (55.6%) and Gw (40.7%), which have been prominent in Japan recently. One isolate was classified in another lineage, which was detected in Japan for the first time, and was similar to a Hong Kong isolate from 2014. Remarkably, the geographic distributions of the 2 major lineages were separated. The Ge viruses were isolated from the main island of Okinawa and the Yaeyama Islands, whereas the Gw isolates were mainly detected from the Miyako Islands. These results suggest that the Ge and Gw mumps viruses mainly caused the mumps epidemics of 2015 in Okinawa, and that they spread independently in separate regions. This is the first report describing the molecular epidemiology of mumps epidemics in Okinawa Prefecture.

Rescue system for measles virus from cloned cDNA driven by vaccinia virus Lister vaccine strain.

A rescue system for measles virus from cloned cDNA was established using CHO/hSLAM cells (Chinese hamster ovary cells expressing a measles virus receptor, signaling lymphocyte activation molecule), LO-T7-1 virus (the Lister vaccine strain of vaccinia virus expressing the T7 RNA polymerase under the control of the early/late p7.5 promoter), and caspase inhibitor. LO-T7-1 drove efficiently the T7 promoter in CHO/hSLAM cells. Rescue efficiency with LO-T7-1 was not as high as that with the vTF7-3 strain based on a neurovirulent vaccinia virus, but was increased by using a caspase inhibitor to block apoptosis of CHO/hSLAM cells induced by LO-T7-1. These modifications resulted in a measles virus rescue efficiency that was even higher than that of previous systems. This safer and more efficient system will facilitate further the genetic manipulation of measles virus in basic research and virus vector development.

Development and biological properties of a new live attenuated mumps vaccine.

To develop a new live attenuated mumps vaccine, a wild mumps Y7 strain isolated from a patient who developed mild parotitis was treated with nitrosoguanidine and ultraviolet, followed by selection of a temperature-sensitive clone. The selected clone, Y125, showed stable temperature-sensitivity in Vero cells. Intraspinal inoculation of marmosets with the Y125 produced only minimal histopathological changes, while intracerebral inoculation of neonatal rats revealed that the Y125 did not cause hydrocephalus. Both these effects of the Y125 were similar to those of the non-neurovirulent Jeryl Lynn strain. Furthermore, subcutaneous inoculation of the Y125 induced high levels of neutralizing antibodies in all Cercopithecus monkeys examined. Although the safety and immunogenicity should be confirmed in further field trials in humans, the present results indicate that the Y125 could be a promising vaccine candidate.

Large-scale preparation of biologically active measles virus haemagglutinin expressed by attenuated vaccinia virus vectors.

A procedure described here allows the efficient and rapid purification of histidine-tagged measles virus haemagglutinin that is synthesized under the control of powerful promoters (PSFJ1-10 and PSFJ2-16) of the highly attenuated vaccinia virus (VV) strain LC16mO. A single affinity chromatography step purifies recombinant haemagglutinin proteins from the lysates of cells infected with the recombinant VVs. The recovery and purity are both very high (a yield of 0.5-2.8 mg/10(8) cells and purity of >94-98%), indicating that this procedure is approximately 400 times more efficient than the conventional methods used to prepare haemagglutinin. The haemagglutinins are correctly transported to the cell surface and have haemadsorption activity. Moreover, the recombinant haemagglutinin proteins cooperate with the measles virus fusion protein to elicit cell fusion activity. In addition, the antibody titres against measles virus, as measured by enzyme-linked immunosorbent assay using the purified haemagglutinin as the capture antigen, correlated closely with neutralization test titres (R(2) = 0.84, p < 0.05), indicating the preservation of immunologically relevant antigenicity. Such recombinant haemagglutinin preparations will be useful in diagnostic tests that measure functional anti-measles immunity and investigate the biological functions and structure of the haemagglutinin.

Vaccinia Virus LC16m8∆ as a Vaccine Vector for Clinical Applications.

The LC16m8 strain of vaccinia virus, the active ingredient in the Japanese smallpox vaccine, was derived from the Lister/Elstree strain. LC16m8 is replication-competent and has been administered to over 100,000 infants and 3,000 adults with no serious adverse reactions. Despite this outstanding safety profile, the occurrence of spontaneously-generated large plaque-forming virulent LC16m8 revertants following passage in cell culture is a major drawback. We identified the gene responsible for the reversion and deleted the gene (B5R) from LC16m8 to derive LC16m8Δ. LC16m8∆ is non-pathogenic in immunodeficient severe combined immunodeficiency (SCID) mice, genetically-stable and does not reverse to a large-plaque phenotype upon passage in cell culture, even under conditions in which most LC16m8 populations are replaced by revertants. Moreover, LC16m8∆ is >500-fold more effective than the non-replicating vaccinia virus (VV), Modified Vaccinia Ankara (MVA), at inducing murine immune responses against pathogenic VV. LC16m8∆, which expresses the SIV gag gene, also induced anti-Gag CD8⁺ T-cells more efficiently than MVA and another non-replicating VV, Dairen I minute-pock variants (DIs). Moreover, LC16m8∆ expressing HIV-1 Env in combination with a Sendai virus vector induced the production of anti-Env antibodies and CD8⁺ T-cells. Thus, the safety and efficacy of LC16m8∆ mean that it represents an outstanding platform for the development of human vaccine vectors.

Combined cytolytic effects of a vaccinia virus encoding a single chain trimer of MHC-I with a Tax-epitope and Tax-specific CTLs on HTLV-I-infected cells in a rat model.

Adult T cell leukemia (ATL) is a malignant lymphoproliferative disease caused by human T cell leukemia virus type I (HTLV-I). To develop an effective therapy against the disease, we have examined the oncolytic ability of an attenuated vaccinia virus (VV), LC16m8Δ (m8Δ), and an HTLV-I Tax-specific cytotoxic T lymphocyte (CTL) line, 4O1/C8, against an HTLV-I-infected rat T cell line, FPM1. Our results demonstrated that m8Δ was able to replicate in and lyse tumorigenic FPM1 cells but was incompetent to injure 4O1/C8 cells, suggesting the preferential cytolytic activity toward tumor cells. To further enhance the cytolysis of HTLV-I-infected cells, we modified m8Δ and obtained m8Δ/RT1AlSCTax180L, which can express a single chain trimer (SCT) of rat major histocompatibility complex class I with a Tax-epitope. Combined treatment with m8Δ/RT1AlSCTax180L and 4O1/C8 increased the cytolysis of FPM1V.EFGFP/8R cells, a CTL-resistant subclone of FPM1, compared with that using 4O1/C8 and m8Δ presenting an unrelated peptide, suggesting that the activation of 4O1/C8 by m8Δ/RT1AlSCTax180L further enhanced the killing of the tumorigenic HTLV-I-infected cells. Our results indicate that combined therapy of oncolytic VVs with SCTs and HTLV-I-specific CTLs may be effective for eradication of HTLV-I-infected cells, which evade from CTL lysis and potentially develop ATL.

Immunogenicity and safety of the vaccinia virus LC16m8Δ vector expressing SIV Gag under a strong or moderate promoter in a recombinant BCG prime-recombinant vaccinia virus boost protocol.

We compared the effect of the very strong pSFJ1-10 and moderately strong p7.5 promoters on the immunogenicity and pathogenicity of the replication-competent vaccinia virus (VV) LC16m8Δ (m8Δ) vector harboring the SIV gag gene in a vaccination regimen consisting of a recombinant BCG-SIVGag (rBCG-SIVGag) prime followed by a recombinant vaccinia boost. m8Δ/pSFJ/SIVGag synthesized more Gag protein than m8Δ/p7.5/SIVGag but replicated less efficiently in vitro. In addition, m8Δ/pSFJ/SIVGag was less pathogenic and elicited Gag-specific IFN-γ(+), CD107a(+), CD8(+) cells more efficiently than m8Δ/p7.5/SIVGag. Vaccination by this regimen elicited long-lasting Gag-specific CD8(+) T cells, the majority of which showed a CCR7(-) phenotype at over 8 weeks post-boost. Tetramer staining analyses revealed maintenance of Gag specific tetramer(+), CD62L(-), CD8(+) T cells for long time in vaccinated mice. However, Gag expression increased the neurotoxicity of the vaccinia vector, indicating the necessity of safety testing for each recombinant VV. We propose that this recombinant BCG prime-m8Δ/pSFJ/HIVGag boost regimen would be a promising vaccination procedure for preventing HIV infection.